Date of Award

1990

Publication Type

Doctoral Thesis

Degree Name

Ph.D.

Department

Chemistry and Biochemistry

Keywords

Chemistry, Biochemistry.

Supervisor

Mutus, B.,

Rights

info:eu-repo/semantics/openAccess

Abstract

Calcineurin (CaN), a calmodulin stimulated protein phosphatase is a heterodimer composed of a 61kD catalytic subunit and a 19kD regulatory subunit. The larger subunit has three binding sites for transition metal ions and also contains the intrinsic metal ions, Zn$\sp{2+}$ and Fe$\sp{3+}$. The smaller subunit contains four "EF-hand" Ca$\sp{2+}$ binding sites. All ten of calcineurin's cysteine residues are found on the catalytic subunit. This study explores the metal ion dependence of CaN using Ca$\sp{2+}$, Mg$\sp{2+}$, Mn$\sp{2+}$ and Ni$\sp{2+}$ as well as its thiol chemistry using Ellman's reagent, iodoacetate, iodoacetamide, glutathione and a novel thiol reagent introduced by this study, 1-p-chlorophenyl-4,4-dimethyl-5-diethylamino-1-penten-3-one hydrobromide (CDDP). CaN was purified from bovine brain using DEAE-cellulose and calmodulin-agarose chromatography. Enzyme kinetics were performed with CaN in the presence of EDTA, Ca$\sp{2+}$, Ca$\sp{2+}$ and Mg$\sp{2+}$, EGTA and Mg$\sp{2+}$, Ca$\sp{2+}$ and Mn$\sp{2+}$ and Ca$\sp{2+}$ and Ni$\sp{2+}$, $\pm$ calmodulin (CaM). The highest activity was observed when CaN was assayed in the presence of Ca$\sp{2+}$, Mg$\sp{2+}$ and CaM. Thiol titrations of CaN revealed the presence of 4 exposed sulfhydryl groups on the native protein, and 9 sulfhydryl groups when the protein was denatured with SDS. Modification of CaN by Ellman's reagent, iodoacetate and iodoacetamide was accompanied by a sharp increase in activity followed by a decrease in activity. This suggests that one or more sulfhydryl groups are essential for the full expression of CaN's activity. Attempts were made to locate the 4 exposed sulfhydryl groups on CaN by titrating the various binding sites with Ellman's reagent in the presence and absence of a blocking group. Free sulfhydryl groups were absent from the CaM binding site as well as from the active site, but titrations of the Mn$\sp{2+}$ binding site in the presence and absence of Mn$\sp{2+}$ revealed the presence of an additional sulfhydryl which may be located in this site. CDDP, a Mannich base derivative of an $\alpha$,$\beta$-unsaturated ketone was found to react selectively with sulfhydryl groups over other groups found in proteins. Further study showed it was unique in that it could react irreversibly with protein thiols but reversibly with low molecular weight thiols.Dept. of Chemistry and Biochemistry. Paper copy at Leddy Library: Theses & Major Papers - Basement, West Bldg. / Call Number: Thesis1990 .W345. Source: Dissertation Abstracts International, Volume: 52-11, Section: B, page: 5810. Adviser: B. Mutus. Thesis (Ph.D.)--University of Windsor (Canada), 1990.

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